Altered Ligand Dissociation Rates in Thyrotropin-Releasing Hormone Receptors Mutated in Glutamine 105 of Transmembrane Helix III

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• 作者：Donato del Camino ; Francisco Barros ; Luis A. Pardo ; and Pilar de la Peñ ; a
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：March 18, 1997
• 年：1997
• 卷：36
• 期：11
• 页码：3308 - 3318
• 全文大小：296K
• 年卷期：v.36,no.11(March 18, 1997)
• ISSN：1520-4995

文摘

Glutamine 105 in the third transmembrane helix of thethyrotropin-releasing hormone receptor(TRH-R) occupies a position equivalent to a conserved negativelycharged residue in receptors for biogenicamines where it acts as counterion interacting with the cationic aminemoiety of the ligand. Maximumlevels of response to TRH in oocytes expressing wild-type TRH-Rs wereindistinguishable from those ofoocytes expressing receptors mutated to Glu, Asn, or Asp in position105. However, the EC₅₀ values foractivation of oocyte responses increased more than 500 times in oocytesexpressing mutant Glu¹⁰⁵ receptors,in which the amido group of Gln¹⁰⁵ has been removed bysite-directed mutagenesis. Charge effects donot seem to be involved in the huge effect of mutatingGln¹⁰⁵ to Glu, since mutation of Gln¹⁰⁵ toAspinduces only a 15-fold increase in EC₅₀. Furthermore,no change in EC₅₀ is observed after mutationofAsn¹¹⁰ to Asp. The affinity shift (identified bychanges in EC₅₀ values for systems of comparableefficacy)in Glu¹⁰⁵ mutant receptors was partially recovered inoocytes expressing Asn¹⁰⁵ mutant receptors.Theseresults and those obtained after substitution of Lys, Leu, Tyr, and Serfor Gln¹⁰⁵ suggest that the presenceand the correct position of the Gln hydrogen bond-donor amido groupare important for normalfunctionality of the receptor. In wild type or Asp¹⁰⁵mutant receptors showing the same maximal responses,decreases in affinity with TRH and methyl-histidyl-TRH correlated withincreased dissociation rates ofhormone from the receptor. Rapid dilution experiments followingsubsecond stimulation indicate thatthe TRH-R is converted rapidly from a form showing fast dissociationkinetics to a form from which thehormone dissociates slowly. Mutation of residue 105 impairs thereceptor shift between these two forms.This effect was demonstrated in a direct way by comparing[³H]methyl-histidyl-TRH dissociation ratesinCOS-7 cells transfected with either wild type or Asp¹⁰⁵mutant TRH-Rs. Thus, residues located intransmembrane helix III positions equivalent to those of thecounterions for biogenic amines, regulatehormone-receptor interactions in the TRH receptor (and perhaps otherreceptors). Furthermore, the natureof the amino acid in these positions may also play a role, directly orindirectly, in conformational changesleading to receptor activation, and hence to signaltransduction.